Novel Mechanisms that Antagonize Hedgehog Signaling at the Cell Surface During Vertebrate Embryogenesis

Abstract

Hedgehog (HH) signaling is a conserved mode of cell-cell communication that is indispensible for embryogenesis. HH proteins are secreted ligands that travel over long distances within a developing tissue to induce distinct cellular responses in a concentration- and time-dependent manner. These properties necessitate precise spatial and temporal control of HH ligand production, distribution, and reception at the cell surface. Failure to properly regulate HH ligands during development produces debilitating congenital disorders. Moreover, deregulated HH pathway activity in adult tissues causes several human diseases. In particular, overactive HH signaling underlies many devastating pediatric and adult cancers. Therefore, understanding the mechanisms that restrain HH signaling will provide key insights into human development and disease pathogenesis. A highly conserved negative feedback loop involving the canonical HH receptor Patched (Ptc in Drosophila; PTCH1 in vertebrates) limits the magnitude and range of HH signaling by binding and sequestering HH ligands. While Ptc up-regulation is essential during Drosophila embryogenesis, PTCH1-feedback inhibition plays only a limited role in mammals. In this dissertation, I have resolved this discrepancy by demonstrating redundant roles between PTCH1 and two additional vertebrate-specific HH-binding antagonists that are induced by HH signaling, PTCH2 and HHIP1. Importantly, I define PTCH1, PTCH2, and HHIP1 as a core network of HH pathway antagonists that play overlapping and essential roles to restrict HH signaling during mammalian embryogenesis. While any single antagonist is largely dispensable for normal development, the combined loss of all three inhibitors yields unrestrained HH pathway activity in the mouse embryo. Moreover, I find that these vital HH antagonists function through distinct molecular mechanisms. In particular, I present evidence to define HHIP1 as a secreted inhibitor of vertebrate HH signaling. Surprisingly, secreted HHIP1 protein is enriched in epithelial basement membranes due to a direct, high affinity interaction with heparan sulfate. Intriguingly, HHIP1 also controls the tissue distribution of endogenous HH ligands within the basement membrane during development. Overall, these data provide novel insights into the mechanisms that restrain vertebrate HH signaling and establish a foundation for further investigation into the role of HH inhibitors during development, tissue homeostasis, and disease processes.